RAT LIVER PHENYLALANINE HYDROXYLASE - A METHOD FOR MEASUREMENT OF ACTIVITY WITH PARTICULAR REFERENCE TO DISTINCTIVE FEATURES OF ENZYME AND PTERIDINE COFACTOR

The activity of rat liver phenylalanine hydroxylase has been measured in systems which, in addition to the components necessary for hydroxylation, contained ascorbic acid and catalase. 6‐Methyl‐tetrahydropterine or the corresponding 6,7‐dimethyl compound was used as tetrahydropterine cofactor. Simple kinetic experiments lead to the determination of rate constants for the following reactions: the aerobic oxidation of the tetrahydropterine cofactor, the reduction of the dihydropterine cofactor by ascorbic acid and other reductants and the conversion of the dihydropterine cofactor from an active to an inactive form. These rate constants are used for the calculation of the actual concentration of tetrahydropterine cofactor in the hydroxylase system and for correction of the measured rates of hydroxylation for the inactivation of the dihydropterine cofactor. Ascorbic acid, as well as other reductants that may be present, increase the instability of phenylalanine hydroxylase, but this effect is negligible at sufficiently high catalase concentrations. Certain non‐reducing inhibitors can also stabilize the enzyme. The initial rate of reaction at 30° is linear during the first 5 or 6 min. In contrast at temperatures of 25° and below there is a lag period, whose duration may be reduced by preincubation of the enzyme with tetrahydropterine cofactor. A similar preincubation of the enzyme with l‐phenylalanine seems to activate the hydroxylase briefly. Copyright © 1969, Wiley Blackwell. All rights reserved